Method and device for rapid homogenisation and mass transport

ABSTRACT

Rapid mixing and homogenisation of reaction mixtures with respect to temperature and chemical concentrations, as well as greatly enhanced mass transport is achieved when the reaction mixture placed in a vassel suitable for centrifugation, and subjected to asymmetric heating, cooling and simultaneous centrifugation at conditions for creating an enhanced flow within the reaction mixture, wherein the flow ensures practically total mixing and homogenisation of the reaction mixture.

The present invention refers to the field of chemistry and biochemistry,and in particular the handling of reaction mixtures in liquid mediawhere a rapid mixing and homogenisation with respect to both temperatureand molecular concentration gradients in the reaction mixture isdesired. It also refers to creating specific flow patterns inside areaction vessel under centrifugation, heating and cooling, as well as tocreating efficient mass transport between the bulk of a liquid and asolid phase, present in said liquid.

BACKGROUND OF THE INVENTION

Many important industrial processes as well as procedures applied inlaboratories of various kinds are dependent on chemical and biochemicalreactions. Commonly the time consumed for completing a process orprocedure is determined by the time it takes for a specific chemicalreaction or reactions to reach equilibrium. This is often referred to asthe kinetic properties of a chemical reaction or simply reactionkinetics. A host of variables influence the reaction kinetics in eachcase, for instance molecular properties and the concentrations ofreactants, temperature, presence of catalytic agents etc.

Typically, increased temperature accelerates chemical reactions byspeeding up key mechanisms like bringing molecules or molecule domainsin contact with each other. Therefore it is common to heat the reactionvessels, for example bringing them in contact with an open flame, hotgas, hot liquid, hot sand or a solid material. This procedure is oftenreferred to as incubation. In biochemical reactions, more sophisticatedprocedures are required to avoid irreversible denaturation of sensitivecomponents upon heating.

One typical problem involved with incubations of fluid reaction mixturesis thermal heterogeneity, because the parts of the reaction mixturebeing in close contact with the walls of the reaction vessel will becomeheated before the more central parts of the reaction mixture.Frequently, there is a risk that part of the reaction mixture becomesoverheated before other parts even reach the desired temperature.Further, in the absence of mixing or agitation, temperature gradientsform in the reaction mixture. Hot subsets of the reaction mixturenormally have lower density than cold subsets, which tend to generatetemperature gradients or discrete layers of more or less isothermalbodies of liquid, so called thermoclines. Thus warm, less dense portionsof the reaction mixture tend to find a position above cold, denserportions. Molecular motion and currents in the reaction mixture willeventually homogenize the reaction mixture with respect to temperature,a process here referred to as temperature homogenisation of the reactionmixture, or homogenisation with respect to the temperature. The time ittakes to homogenise a reaction mixture with respect to the temperaturemay contribute substantially to the time required for the completereaction.

However, time-consumption in itself is not the sole problem involvedwith temperature homogenisation of chemical reaction mixtures. Incertain incubation procedures such as the repetitive temperatureadjustments involved in so called thermocycling processes, e.g. forperforming polymerase chain reactions, also known as PCR-reactions, longtemperature homogenisation periods favour unwanted side-reactions,sometimes causing severe quality problems with respect to theaccuracy-and specificity of the obtained PCR-product.

In an alternative amplification process, known as rolling-circleamplification (RCA), the thermocycling is replaced by one singletemperature adjustment, followed by a prolonged incubation. In thisapplication, it is important that the desired temperature is reachedrapidly and with high accuracy within the entire sample volume, in orderto avoid unspecific onset of the amplification process, and theformation of products, which will remain and be amplified during theincubation.

In the ongoing strive to miniaturize chemical reaction volumes, asevident e.g. in the field of high throughput screening (HTS),combinatorial chemistry etc., several other problems are encountered. Ina small reaction vessel, such as a small test tube or a well on amicrotitre plate, both the mixing and temperature homogenisation ofsample and reagents may become severely restricted. When two or moremiscible fluids are mixed, we normally assume that they first form ahomogenous mixture, which then reacts. This is however rarely the case.

Assays for concentration determinations have a wide range of formats andconfiguration. Quite a few are based on solid phase immobilisation ofone component in the binding assay and determination of the amount ofanalyte that can be detected on the particular surface. Of particularinterest for all assay formats, and in particular solid phase assays, isa proper homogenisation and efficient mass transfer from a large volumeto a defined ligand on the surface. With an increased focus onmultiassays, an increased interest has been focused on array formatsthat effectively can analyse low concentrations of many analytes fromone defined sample volume.

Conventional microtitre plates and cuvettes are often manufactured frompolystyrene, a hydrophilic polymer. Without dwelling on the exactbehaviour of the liquid at the vessel boundaries, it can be concludedthat stagnant areas will form and insufficient mixing easily occur in asmall reaction vessel, such as a well on a microtitre plate. Theproperties of the reactants and sample fluids also influence theirinteraction with each other and with the vessel boundaries. Partialsegregation, the formation of layers, aggregation and so on, are only afew examples of irregularities that can be encountered in a reactionvessel.

There are reasons for distinguishing between two different phenomenacausing problems with heterogeneous temperature distribution in areaction mixture. The phenomena caused by the interaction between thefluid and the walls, appearing close to the walls of a reaction vesselis a problem, which increases when reaction scale decreases. Incontrast, the phenomena involving central parts of the liquid body beingcolder than the liquid close to walls when heating a reaction vesselfrom the outside, increases when reaction scale increases. This is thereason why thermocycling devices for use in processes in which propertemperature homogenisation is required (e.g. processes like PCR), have avery narrow dynamic range with respect to the reaction scale as thesurface to volume ratio has to be high. Typically, in PCR-reactionsthese problems are most severe when the reaction volumes are less than 5μL or larger than 50 μL.

Another problem, seemingly unrelated to the mixing and temperaturehomogenisation issues, is that of evaporation. In order to minimizeevaporation, there is a tendency that the reaction vessels, inparticular the wells on microtitre plates, are made both deeper and morenarrow. Naturally, this further enhances the previously mentionedproblems of insufficient mixing and temperature homogenisation.

So far, temperature heterogeneity has been discussed in terms ofproperties in a single reaction vessel. Especially when discussingminiaturisation of multi-sample or parallel applications, such asassays, different applications in combinatorial chemistry, chemicalsynthesis, and HTS etc., yet another dimension of temperatureheterogeneity needs to be considered; that of variation betweenindividual reaction vessels. In assays with comparative purposes (i.e.with or without quantitative analysis like screening for novel drugcandidates, mutations in nucleic acids, single nucleotide polymorphismand so forth) it is important to consider the reproducibility, commonlyreferred to as well-to-well uniformity.

Since the mechanisms behind poor thermal uniformity are difficult todescribe and simulate accurately, the only available solution to theproblem is often to focus on means to enhance the homogenisationprocesses. To do this, various strategies are applied. Mechanicalagitation is perhaps the most commonly employed method, this agitationincluding both stirrers in the reaction mixture, as well as agitation ofthe entire reaction vessel. Ultrasound is another often used method toperform agitation and still another method is to force the reactionmixture to pass a defined area repeatedly, e.g. by pumping the reactionmixture through a fluid channel or cell, in which reagents or analytesare immobilised.

The mass transport of chemical reagents or biochemical components in thevolume is of vital importance to achieve reproducibility and uniformconditions in the volume. Mass transport is also of vital importance insolid phase assays or synthetic situations, where material has to betransferred from the bulk of a fluid to a solid surface over thediffusion limited stagnant layer of said fluid. This is a limitingfactor for speed and sensitivity of most ligand binding assays.

The problems underlying the invention can be easily derived from thestate of the art, considering the above introduction read with theknowledge of a person skilled in the art.

PRIOR ART

WO 98/49340 (PCT/AU98/00277) discloses a temperature cycling device andmethod where a reaction mixture and a sample is loaded into loadingwells on a disposable rotor, which rotor is then placed into acentrifugal thermal cycling device and spun, so that the reactionmixture and sample are moved by centrifugal force to a reaction well atthe periphery of the rotor. The device comprises heating means, forexample infrared lights, convection heating elements or microwavesources. Interestingly, also provisions for cooling the rotor areincluded in the specification. According to one embodiment, the rotorspeed is increased, resulting in air being drawn into the device andrapidly cooling the contents of the reaction chambers at the peripheryof the rotor. In addition to ambient air, a coolant gas can be used.Refrigerated air is given as an example of coolant gases. Importantly,the disclosure of WO 98/49340 implies the use of different speeds ofrotation. Further, WO 98/49340 does not address the problems of mixingand homogenous temperature. For example, it does not specify thedirection of heating, nor does it contemplate simultaneous heating andcooling.

DE 19501105 A1 discloses a centrifuge with a temperature control systemwhere a circulating fluid enters the rotor from above and flows outwardsand downwards in the direction of the radius, around the test tubes orsample containers. The inventor of the centrifuge according to DE19501105 criticises the hitherto known devices using a radiating sourceof heat and rejects them as unsatisfactory.

WO 00/58013 of the present applicant describes a method and device forsimultaneous centrifugation and heating and optionally cooling ofsamples.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that controlled and highlyeffective mixing and homogenisation, both with regard to temperature andto molecular concentrations, is achieved when the reaction mixtureplaced in a vessel suitable for centrifugation, and subjected toasymmetric heating, cooling and simultaneous centrifugation atconditions for creating a controlled flow within said reaction mixture,wherein said flow ensures practically total mixing and homogenisation ofthe reaction mixture.

One important advantage of the rapid mixing and homogenisation of thepresent invention is that it is non-invasive in the sense that noimpellers, stirrers or other devices need to be brought in contact withthe reaction mixture. Another important advantage is that the rapidmixing and homogenisation appears to be independent of the reactionvolume, that is the desired result is achieved both in microscopic andmacroscopic reaction volumes.

Another important aspect of the invention is the unexpected flow patternof liquid in the vessel and the high linear flow rate. A laminar flow iscreated in close proximity to the surface of the vial and this willconsiderably improve the mass transport from the bulk volume to thesurface.

Of particular interest is a special section of the vessel, said sectiondefined by the rotation, where the fluid flows in the direction of theg-force, and where the total volume of the fluid will pass repeatedlyover a limited surface area during the centrifugation period.Consequently, high mass transport is favoured, in combination with ahigh degree of recirculation over a defined area.

The present invention makes available an improved reaction vesselaccording to the attached claims, incorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described in closer detail in the followingdescription, examples, and attached drawings, in which

FIG. 1 schematically illustrates the flow in a reaction vessel 1 duringthe conditions created according to the present invention(superconvection). The direction of the gravitational force, create bythe centrifugation; is indicated by the arrow “g”. The upward flow inthe reaction vessel is illustrated by the light arrows A, and thesurface-oriented flow by the filled arrows B.

FIG. 2 shows the reaction vessel of FIG. 1 from above, the upward andsurface-oriented flow being the same, illustrated by arrows A and B,respectively. The direction of the gravitational force is againindicated by “g”, now pointing into the plane. Additionally, a downwardflow or a local “sink” is indicated as C in contact with the vesselsurface. Further, the arrow “v” indicates the direction of rotationduring centrifugation.

FIG. 3 a shows an embodiment of the invention, where a reaction vessel 1is designed to take advantage of the flow pattern achieved. Local means,capable of interaction with at least one component of the reactionmixture, are positioned as discrete dots or spots 2, 3, 4 and 5 in thearea of the sink.

FIG. 3 b shows another embodiment, where a reaction vessel 1 has localmeans 6, 7, and 8 positioned in the shape of elongated areasintersecting with the sink.

FIG. 3 c shows a third embodiment, where said local means, here shown as10, 11, 12, and 13, are positioned on an insert 9, preferably adapted tothe geometry of the reaction vessel 1 and insertable therein so thatsaid means will become positioned in the area of the sink.

FIG. 4 illustrates in the form of an example, how a reaction vessel 1can be designed having specific geometrical features, 14 correspondingto features of a rotor 16, so that the correct orientation of theindividual vessels can be ensured.

DESCRIPTION

In the following description of the invention, certain definitions willbe used. They are to be interpreted as outlined below:

The terms “gravitation”, “gravitational field” and “direction of thegravitational field” are described with vectors, the direction of thegravitation field being the same as the resulting vector when a vectorrepresenting the centrifugal force, that is a vector at right angle tothe axis of the centrifuge rotor directed from the centre of the rotor,is added to the vector representing the earth gravity. Consequently,downwards is in the present text defined as the same direction as thegravitation field as defined by the summed vectors representingcentrifugal force and gravitation.

The term “reaction mixture” means any fluid reaction mixture, preferablya liquid reaction mixture, in which the reaction kinetics is influencedby temperature and where a faster, more efficient and homogenoustemperature adjustment is desired. A non-exhaustive list of examples ofreactions, suitable for the present device and method arechemical/biochemical reactions within the field of chemical synthesis,combinatorial chemistry, high throughput screening, assays, methods forthe determination of the presence of or the concentration of a givensubstance, and biochemical reactions involving incubation or temperatureadjustments, e.g. repeated temperature adjustments, cyclic temperaturechanges, including a polymerase chain reaction (PCR), a ligase chainreaction (LCR), a “gapped-LCR-reaction”, a nucleic acid sequence-basedamplification (NASBA), a self-sustained replication (3SR), atranscription mediated amplification (TMA), a stand displacementamplification (SDA), a target amplification, a signal amplification,rolling-circle amplification (RCA), or a combination of any of theabove.

Typically a polymerase chain reaction involves the following steps:

1) Preparation of the reaction mixtures, i.e. preparation of the samplesto be tested;

2) Amplification, i.e. the exponential replication of the DNA molecules;and

3) Detection of specific sequences for example by electrophoresis orhybridisation.

This outlined principle is applicable, mutatis mutandis, to many of theabove reactions. In conventional PCR, step 2) involves repeatedtemperature changes to take the reaction mixture through the steps ofannealing and extension of the nucleotide strands. Inefficienttemperature homogenisation, e.g. diffuse temperatures and temperaturegradients in the reaction mixture, leads to unspecific amplificationproducts. The necessity of a fast and homogenisation with respect totemperature is central for the quality and reliability of the reaction.Further, an effective mass transport within the sample, and betweencomponents thereof, is equally important.

The term “assay” in the above encompasses all forms of assays, such ashomogenous and heterogeneous assays, binding assays, solid phase assays,particular examples including—but not limited to—ELISA assays,immunoassays, enzyme-based assays, non-labelled assays, affinity assays,fluorescence assays etc.

The term “reaction-vessel” means any vessel capable of containing areaction mixture within a temperature range necessary for performing thedesired reaction/reactions. Examples of reaction vessels suitable foruse according to the invention include, but are not limited to, thefollowing: test tubes, so called micro tubes, Eppendorf-tubes, a singlewell or a multitude of wells in a microtitre plate, such as a microtitreplate of the 96-hole format, and various formats with a high densityarrays of wells, such as the 192-hole format, the 384-hole format,denser formats or the like. Further examples of reaction vessels includeflow channels, cells and volumes, defined in a device such as disc orother device, suitable for rotating, e.g. the analysis platformcorresponding to the conventional CD-format. The reaction vessel for useaccording to the present invention can be a conventional, commerciallyobtainable reaction vessel as listed above, or a reaction vesselspecially adapted for use according to the invention.

The term “asymmetric” as in “asymmetric heating and cooling” refers toheating and cooling acting only on subsets of the reaction volume in areaction vessel.

The terms “controlled flow” and “enhanced flow” are used to define thenon-intuitive flow created in a reaction vessel according to the presentinvention. A flow is controlled or enhanced when it differs from theflow normally present (if any) in a reaction vessel subjected to eitherto heating only, cooling only, or centrifugation only. A flow is alsoheld to be controlled or enhanced when said flow, it direction, speed,orientation or other properties are deliberately influenced.

The present inventors have surprisingly found that extremely rapidmixing can be achieved when a reaction mixture is subjected tosimultaneous heating and cooling during the influence of a centrifugalforce. This extremely rapid mixing is tentatively calledsuperconvection. Importantly, it has been shown that this mixing isachieved already at small temperature differences within the reactionmixture.

The rapid mixing, mass transport and temperature homogenisation,achieved under the conditions developed by the inventors, is driven bychanges in density in the liquid based on asymmetric heating/coolingeffects and the Coriolis' effect due to rotation of the entire volumeduring the centrifugation. This in combination give rises to extremelyhigh liquid streaming which in turn homogenises the reaction mixtureboth with respect to temperature and differences in substanceconcentrations within the entire vessel volume. The present inventioncreates a non-intuitive flow along the side walls of the tube in thinlayers and a steep channel of liquid flow in the direction of theg-forces in a discrete position in the reaction vessel, said positionbeing defined by the direction of the rotation. This will be called “thesink” in the following description and examples. Surprisingly, the flowalong the sidewall appears to first take place horizontally, on bothsides of the vessel, until it reaches the sink, where it turns downward.The flow pattern achieved according to the present invention isschematically shown in FIG. 1 and 2.

It is very surprising to note, that the surface-oriented flow takes theform of extremely thin layers. This has particular advantages. Thinlayer flow of liquid gives new properties to the system in relation tomixing of the volume. The mass transfer characteristics for a thin-layercell are well known, and it has now been shown by the present inventorsthat superconvection involves a flow of liquid, similar to that of athin-layer cell, occurring along the cell walls. However, the linearflow rate has been estimated to be in the range of m/s according totheoretical calculations dependent on temperature difference andcentrifugation speed. This very surprising finding should be comparedwith the results obtained with conventional flow-based instruments,where at a normal flow rate of 15 microliter per min the linear flowrate is in the order of 10 mm/s in a 50 micro-meter high flow cell. Thisshows that superconvection as achieved by the present invention resultsin 10 to 100 fold higher flow rates. Consequently, the mass transfer ismuch more effective than pumping liquid through a cell for surfaceuptake or liquid transport by electroendosmotic effects.

As superconvection according to the present invention takes place in areaction vessel with a fixed volume, it follows that the entire volumewill repeatedly pass the surface area and the total transfer of materialfrom the volume to the surface becomes very effective. This is animportant advantage not least for highly sensitive assays where theconcentration of analyte is very low.

One aspect of the present invention is to take advantage of this flowpattern by positioning means capable of interaction with at least onecomponent of the reaction mixture at locations, determined by said flowpattern. According to this aspect of the invention, said means arepositioned in the area of the sink, or intersecting the downward flow insaid area. See FIGS. 3 a and 3 b. Another embodiment involves thepositioning of said means, capable of interaction with at least onecomponent of the reaction mixture, on an insert, suitable for insertionin the reaction vessel. See FIG. 3 c. When designing such inserts, andoptionally also the reaction vessels that receive them, care should betaken to ensure that the final orientation of said insert in thereaction vessel corresponds to that of the sink.

According to one embodiment of the above aspect of the invention, saidmeans capable of interaction with at least one component of the reactionmixture are small surface areas for adsorption of analyte from theliquid. As the entire volume will pass over a specific surface areaseveral times, it is possible to immobilise a ligand on a specific, verysmall part of the surface, either on the walls or in the particular areawhich effectively will work as a sink. In many assay configurations itis important to transfer as much analyte as possible from the volume toa small surface area to reduce noise from background in the detectionstep. The volume that passes over a small spot during the analysis oftenlimits this. The effective mass transport of analyte from the solutionto the surface for adsorption becomes a limiting factor.

According to another embodiment of this aspect of the invention, saidmeans capable of interaction with at least one component of the reactionmixture, are means for facilitating the detection of a component or thedetermination of a property of the reaction mixture. Reaction vesselshaving such means are then used together with detection methods thatfocus on selected areas inside the vessel, such as confocal microscopyor any other related or equivalent technology. High surfaceconcentration in small spots will facilitate the detection.

According to another embodiment of this aspect of the invention, saidmeans capable of interaction with at least one component of the reactionmixture, are means having structured surfaces where each surface entityhas one analyte specificity. In this way addressing and multi-analysisin one vessel can take place, even when the chemical environment is thesame under all assay conditions. The sink part of the reaction vessel isin this aspect a point for orientation of the tube in the used assaywhere conditions are different regarding flow properties in comparisonwith the rest of the vessel. This is the place where all liquid from thebulk passes as a thin stream of liquid several times. The size of themeans capable of interaction with at least one component of the reactionmixture, here the analyte specific areas, can vary considerably. Thisdepends on many different aspects, including the nature of the detectionstep performed after the effective superconvection controlled adsorptionhas taken place. The detection and subsequent steps do not necessarilyhave to be performed under centrifugation as long as the analyte hasbeen concentrated to a small area, e.g. on patterned surfaces, duringthe rapid mixing and homogenisation achieved by the present invention.The rapid mixing and homogenisation according to the invention may beonly one step of the full assay procedure.

This aspect of the invention gives the user the freedom to address thesink part with a series of analyte specific small areas or arrays,knowing that the entire volume of reaction mixture will pass these areaswith the maximal speed. These means or here surface areas that can beaddressed can be as small as the detection and addressing technologyallows. This embodiment has the advantage of improving the sensitivityof an assay, by adsorbing a low concentration from a large volume ofsample to a very small surface area. Another aspect is to use the sameanalyte specificity in the sink part as well as other parts of the wallwhere the flow rate is different. This will give different mass transferproperties and thus cover different part of the assay concentrationrange.

Another embodiment based on this aspect of the invention is thepossibility to create arrays of particles, reagents etc. in differentlocations on the surface of the vessel. These can, as discussed above,be located in the area of the sink, or in the vicinity of said area.These arrays, or individual spots or dots or areas having particularproperties, can also be located at other places on the inner surface ofthe reaction vessel, based on knowledge of the flow pattern and thedesired interaction between said arrays, dots, spots or areas, and thereaction mixture.

Still another aspect of the present invention, based on the high flowrates encountered in thin layers close to the surface of the reactionvessel, is to create a pattern on the inner surface of the reactionvessel in such a way that the laminar flow is disturbed. According tohis, the means capable of interaction with at least one component of thereaction mixture are means interacting physically or mechanically withthe flow. The mass transport under such conditions can be varied asdesired, dependent on the centrifugation speed and flow characteristicsclose to the surface and the pattern on the surface. Such flowdisturbances induced on the surface can be combined with chemicalpatterns to induce optimal mass transfer to particular areas on thesurface, for example areas to which defined ligands are immobilised.According to an embodiment of this aspect of the invention, the flowcharacteristics can be influenced, either in the reaction vessel as awhole, or in selected areas thereof, by adding specific topographicalfeatures to the inner surface, said features interfering with thelaminar, surface-oriented flow. Said features can be arranged randomly,in a specific area, but are preferably arranged in the form of anordered array.

In addition to the above, said means capable of interaction with acomponent or property of the reaction mixture, can be means chosen amongmechanical means interacting with the flow, such as means guiding theflow, causing turbulence etc, means for transferring heat, means forguiding light or radiation, arrays or particles or substances, definedareas, dots or spots with a chemical or biochemical component whichinteracts with a component in the reaction mixture etc.

The present invention can be applied to chemical synthesis. Chemicalsynthesis is a major bottleneck in the search for new drug candidates inpharmaceutical development. Rapid synthesis and reduction of impuritiesare important aspects as well as the parallel processing of synthesis.This finds application for example in HTS and in combinatorialchemistry. One method of chemical synthesis involves microwave heatingand magnetic stirring. The heating process is effective but the magneticstirring is unsatisfactory, as it gives rise to temperature gradientsinside in the vessels, e.g. so called hot spots. This is valid both forthe heating phase but even more so for the cooling phase. Thetemperatures vary, but temperatures up to 200° C. are used and thesynthesis may take place under high pressure in many different solvents.

Another aspect of chemical synthesis is the rapid cooling, necessary toavoid side reactions after the main product has been formed. In somesituations, there is a need to cool the reaction while it takes place,although this is less frequent. The rapid heating and cooling procedurescan be of vital importance for the outcome of the reaction. However,with the presently available technology, the stirring and mixingprocesses become limiting factors. According to one leading actor inthis field, the time to reach working temperature was 50 s for areaction time of 30 s at 140° C. Correspondingly, about 70 s wasrequired to cool the same reaction mixture to about 50° C. Microwavetechnology has taken traditional chemical synthesis into a new era.However, it has not solved the important aspect of temperature gradientsformed during heating and especially during cooling, which seems to bepassive diffusion of heat over relatively long distances in combinationwith magnetic stirring. The microwave absorption characteristics arenotoriously difficult to control, but modern synthesis instrumentationcan give a much better distribution of heat than traditional microwavetechnology. Still the temperature in the reaction mixture is frequentlyinhomogeneous. Further, it is clear that it still takes relatively longto approach the desired reaction temperature, particularly as it isimportant not to exceed the same. As a result, the time to reach adesired temperature is much longer than what would be optimal from thepoint of controlled synthesis. Also the cooling is inefficient, and thetime needed to reach a reasonable temperature is 2-3 times longer thanthe reaction time at the desired temperature. It is easily seen thatsuch conditions are less than optimal.

According to one aspect of the present invention, the rapid mixing andhomogenisation according to the invention is used to reduce the time forheating and cooling in chemical synthesis applications. This willdramatically decrease the time for a complete synthesis. Further, theamount of impurities formed at non-optimal temperature levels becomeslower due to the rapid mixing and homogenisation with regard totemperature. This is a considerable and surprising advantage of thepresent invention. Further, the size of the reaction volume can bevaried within a large interval, both up as well as down, towards themicroliter range. In particular the microliter to sub microliter rangeis of interest when it is desired to reduce the amount of chemicalsolvents and reagents.

The method according to the present invention will also increase themass transport of chemical reagent in the synthesis volume, as therewill be effective mixing also under the reaction period at desiredtemperature due to the asymmetric heating. This is a surprising aspectof the invention, as it has been shown that the rapid mixing andhomogenisation takes places already at very small temperaturedifferences in the reaction mixture. This is valuable in applications,where the requirements for temperature accuracy are high, and inparticular in such applications where a given temperature limit may notbe exceed.

There are several examples in the literature on microwave assistedchemical synthesis and one overview can be Larhed, M., Hallberg, A. DrugDiscovery Today 2001, 6, 385-395. It is conceived that the inventivemethod can be applied to improve any of these. The present inventionmakes available methods and devices for use in these applications.

One particular embodiment of the above aspect of the invention relatesto solid phase chemical synthesis. One advantage of the inventive methodis the distribution of flow along the surface of the reaction vessel inthe form of a parallel laminar flow. This results in an effective masstransfer of substance between the bulk and the surface. This can also beused e.g. for solid phase synthesis, which is routinely used forsynthesis of peptide and nucleic acids. Traditionally such synthesismethods are based on the use of beads in order to improve surface tovolume properties. However, for analytical applications it is often moreconvenient to apply the synthesis in the same reaction vessel as wherethe analysis takes place. Sequential steps with alternating washing andreagent addition are necessary to build polymers on the surface of thevessel. According to the invention, the rapid mixing and homogenisationachieved by simultaneous, asymmetric heating and cooling is applied tosolid phase synthesis.

A centrifugation based method and/or system has the added advantage thatreagent cartridges, such as CAPILETTE® (ALPHAHELIX AB, Uppsala, Sweden)can be used for reagent addition. When the reactions are sequential,washing steps have to be introduced. In some situations an effectiveimmobilisation of ready-made polymers can be a better alternative, andto this end, the application of the inventive method of rapid mixing andhomogenisation and in particular the surface-oriented rapid flow will beadvantageous.

Another aspect of the present invention relates to homogenousimmunoassays, and the present invention makes available methods anddevices for such assays. In this context, the term “device” includesboth platforms for the performance of such assays, as well as componentsthereof, reaction vessels, multi-sample plates, and devices handlingsuch components, as well as kits comprising such components andreagents.

Consequently, another aspect of the rapid and effective mixing accordingto the present invention is the application in assays, such as bindingassays, solid phase assays, immunoassays or any other receptor basedassay. In this context, the term receptor based assay comprises assaysbased on all types of receptors, such as membrane bound receptors,soluble receptors, lectins, antibodies, fragments of antibodies,peptides, and synthetic receptor molecules.

The present invention also finds utility in rapid temperatureinactivation in assays and synthetic processes, and the presentinvention makes available methods and devices for such assays andprocesses. In PCR and PCR-related applications, it is a major benefit iftemperature ramping could be performed more quickly and accurately thanpresently is the case. The present invention when applied here results nconsiderable improvements. It should be noted, that in PCR, the increasein temperature is used as a step for denaturation of the DNA. Thisdenaturation effect can be used in several other applications, where onecomponent has to find its maximum activity or where components need tobe inactivated. Compared to nucleic acids, enzymes and other proteinsoften have a relatively low temperature optimum for activity before theyare inactivate by thermal denaturation. In many assay procedures, acomponent (e.g. proteases) needs to be inactivated before the additionof reagents. Heat inactivation of components in the sample is often usedbefore performing the assay, and complement inactivation in serum is astandard procedure. The present invention can advantageously be appliedto the above steps.

According to one embodiment of the above aspect of the presentinvention, heat inactivation of components in reaction-mixtures can beintegrated in one and the same procedure using the rapid mixing andhomogenisation achieved by asymmetric heating and cooling duringcentrifugation. This has considerable advantages, as it will reduce timeand the amount of handling steps in the assay. Heat inactivation usingthe invention can also be used to stop the activity of added componentsin the assay, and by using different systems such as heat stable or heatlabile components from various sources, the inactivation can also beperformed in sequence with increased stability and denaturationtemperature. Examples of this can be heat labile UTNG in RT-PCR assaysto perform one tube amplification.

The present invention is also advantageously applied to the field ofsolid phase assays and the present invention makes available methods anddevices for such assays. In this context, the term “device” includesboth platforms for the performance of such assays, as well as componentsthereof, reaction vessels, multi-sample plates, and devices handlingsuch components, as well as kits comprising such components.

Solid phase immunoassays or any other receptor-based assays are eitherof competitive or sandwich type. There is a trend towards morehomogenous assays as they are more effective with respect to mixing andmass transport than solid phase assays. However, solid phase assaysoften give higher sensitivity, better resolution and largerconcentration range in the analytical procedures. One particular problemof solid phase assays is the mixing and mass transport of reagents andanalytes to the surface of the vial. The application of the presentinvention to such assays will reduce the time for incubation of solidphase assays of competitive or sandwich type, due to the rapid flowalong the walls of the reaction vessel. Another advantage will begreatly increased sensitivity of the assay.

According to one embodiment of the above aspect of the invention, onecomponent in the assay is immobilised on the wall of the reactionvessel. The rapid mass transport of analyte to the surface achieved bythe present invention will reduce the time needed for the assay. A largeconcentration range can be obtained, as the measurements can beperformed on the walls and on the sink part of the reaction vessel wheremass transport is different. It is possible to vary not only theaffinity and other properties of the binding molecule, but also to varysurface concentration of both binding molecule and flow properties inthe same vessel during the same assay procedure.

In sandwich assays, the reagents are added in series separated bywashing procedures. It is also possible to use the rapid mixing andhomogenisation according to the invention as well as a heating step toinactivate molecules in an assay as long as other components havedifferent temperature stability. In a centrifugation based system,reagent cartridges, such as CAPILETTE® (ALPHAHELIX AB, Uppsala, Sweden)can be used to distribute new reagents at a desired time andtemperature.

As a general aspect of the present invention, it becomes possible todesign devices for performing reactions in fluid media, where the rapidmixing and homogenisation achieved by asymmetric heating and coolingduring centrifugation, as well as the surprising flow pattern inside thereaction vessel plays a role. Previously in this description, reactionvessels have been mentioned. Such reaction vessels are, according to theinvention, designed to make use of the flow pattern, or example bydesigning the entire shape of the reaction vessel in such manner as toemphasis or take advantage of said flow pattern. It is also conceivedthat the reaction vessel is designed so as to facilitate or increase theeffects of asymmetric heating or cooling. Examples include vesselshaving internal or external means interacting with means for heatingand/or cooling, e.g. external fins dissipating the heat from thereaction vessel and increasing the cooling effect on the rotatingvessels. Another embodiment, also dwelled upon previously in thedescription, includes internal means, capable of interaction with atleast one component of the reaction mixture, positioned inside thereaction vessel, in locations determined by the flow pattern. Apreferred location is in the area of the sink, as the entire volume ofthe reaction mixture will pass here during the rapid mixing andhomogenisation. Further, the reaction vessel itself may be provided witha particular shape or added features, corresponding to a shape orfeatures of the rotor of a device capable of asymmetric heating, coolingand centrifugation, the shape or feature ensuring the correctpositioning of the reaction vessel in said rotor with respect to thedirection of rotation, as well as the direction of the gravitationalforce during centrifugation.

A particular embodiment of the invention is a combination of a vesselhaving an external feature in the shape of a longitudinal edge, or anedge, notch or similar, said feature corresponding to a feature of thehole in the rotor or part thereof, receiving and holding said vessel.The presence of said features ensures that each vessel is correctlyorientated in the rotor, with respect to the direction of rotation andthe direction of the g-force during centrifugation. Optionally, saidfeatures are such, that only vessels having a particular feature, e.g. anotch, will fit in the holes in the rotor, e.g. holes having acorresponding constriction. Another option is such, that the features ofthe holes in the rotor are such, that vessels requiring a pre-definedposition, will only be possible to insert in such position, whilevessels not requiring a particular positioning, can be freely inserted.This embodiment is illustrated in FIG. 4, where the longitudinal edge 14corresponds to a notch 15, in each hole in a rotor 16.

Another embodiment of the invention is a vessel having internalfeatures, influencing or interacting with the flow in the vessel. Thisembodiment is also illustrated in FIG. 3 a through c, where the one ormore of the features 2, 3, 4, 5, 6, and 8 on the inner surface of thevessel 1, as well as features 10, 11, 12, or 13 on the surface of aninsert 9, suitable for positioning in the vessel 1, is such a feature.Examples of topographical features on the inside of the vessel,interacting with the flow, include negative features, such asindentations, dimples, dents, scratches, notches etc, or positivefeatures, such as points, dots, specks, corrugated areas, mattedsurfaces etc. According to a specific embodiment, said positive ornegative features are combined with immobilised reagents, substrates orother chemical properties, designed to maximise mass transport betweensaid reagents etc and the bulk of the liquid in the vessel during theflow conditions created according to the present invention.

The present invention also makes available a device for performing therapid mixing and homogenisation, as well as for creating the flowpattern within the reaction vessel. Such device requires means forholding at least one but preferably a multitude of reaction vessels, andmeans for subjecting these to centrifugation. Preferably such meansconsist of a rotor, e.g. a rotor chosen among the following: a drumrotor, a swing-bucket rotor and a fixed angle rotor.

Further, said device requires means for creating a temperaturedifference within the reaction vessel or vessels, preferablyasymmetrically heating the contents of, and most preferably heating asub-portion of said contents, preferably the central portion of thereaction mixture. Said means can be any heating source, capable ofheating the contents and preferably a subset of the contents of thereaction vessels, e.g. a radiating source, such as a heating elementwith electric resistance wires, an IR-source, a microwave element andthe like. Preferably said heating means are heating means capable offocusing the heat to a sub-portion of the reaction mixture, such as thecentral portion thereof.

Further, said device requires means for cooling the outside of thereaction vessel. The cooling source or means for cooling can be chosenamong convection cooling and a circulating cooling medium, e.g. arefrigerated gas, such as air and preferably nitrogen. In an embodiment,such as those shown in the attached drawings, the cooling medium is letinto the mantle and thus comes in contact with the rotating reactionvessels. According to another embodiment, the environment of the rotoris refrigerated with exception of the mantle. By moving the mantle inrelation to the rotor, e.g. raising or lowering it into close proximityof the rotating reaction vessels, said vessels are heated. By lowering,raising or otherwise removing the mantle, the surrounding coldenvironment is again allowed to contact the rotating reaction vessels.Instead of moving the mantle, the rotor can be moved while the mantle iskept at a fixed position.

Further, the device preferably includes means for temperaturemeasurement. With an IR sensor or other rapid sensor, an on-linemeasurement of temperature is obtained. When using fact that the rapidmixing and homogenisation is achieved also at very small temperaturedifferences in the reaction mixture, the need for fast and accuratetemperature measurement is reduced. When using higher temperatures, theneed for fast and accurate temperature measurement is greater.

EXAMPLES

In preliminary studies, microcentrifuge tubes having a volume of 50 μlwere centrifuged at a speed of 10,000 rpm. The sample had an initialtemperature of 90° C. and the gas surrounding the rotor was kept at 10°C. The temperature difference was approximated to be about 75° C., whenthe properties of the thermoplastic microcentrifuge tube had been takeninto account. Under these conditions, it was found that completehomogenisation was achieved in fractions of a second. Theoreticalcalculations indicate, that flow velocities in the excess of 1 toapproximately 1.5. m/s are reached in thin fluid layers. This is asurprisingly high velocity, and based on this, it can be shown that thehomogenisation with respect to temperature, concentration etc in thevolume of the vessel, as well as the mass transfer between the bulk ofsaid vessel and its surface will be greatly enhanced.

Further calculations show that the flow velocity is linearly dependenton the temperature difference, indicating that the rapid homogenisationand mass transport will be achieved also at lower temperaturedifferences. It is also estimated that the inventive flow pattern isachieved in a very wide volume interval, ranging from extremely smallvolumes such as volumes enclosed in vessels, compartments and channelshaving a diameter of about 10 μm and upwards, as well as in largervolumes, and even volumes in the order of about 10 ml and more. The flowpattern is however believed to enter a more turbulent stage withincreasing vessel volume. Preferably the inventive homogenisation andmass transport is applied to reaction mixtures in volumes in theinterval of about 1 μl to 1 ml, preferably about 10 μl to about 500 μl,and most preferably 50 μl to about 150 μl.

With regard to speed of centrifugation, it is estimated that theinventive homogenisation and mass transport is achieved already atrotational speeds about 1000 rpm, whereas a practical interval is heldto be about 5,000 to about 15,000 rpm, preferably about 6,000 to about12,000 rpm.

At lower speeds, the effectiveness of the inventive homogenisation andmass transport is reduced. The same is believed to be the case for smallvolumes, where the properties of the liquid in question, as well as thegeometry of the vessel will become limiting factors. It is howeverconceived, that there are applications, where even a lover degree of theinventive homogenisation and mass transport will represent animprovement over existing methods.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionwhich is set forth in the claims appended hereto.

1. A method for rapid homogenisation of reaction mixtures with respectto temperature and chemical concentration, the reaction mixture beingplaced in a vessel suitable for centrifugation, and subjected toasymmetric heating, cooling and simultaneous centrifugation,characterized in that said asymmetric heating and cooling consists inthe creation of a temperature difference between subsets of saidreaction mixture, and the centrifugation is performed at conditionssufficient for creating an enhanced flow within said reaction mixture,wherein said flow ensures practically total mixing and homogenisation ofthe reaction mixture.
 2. The method according to claim 1, wherein theasymmetric heating and cooling consists of the walls of the reactionvessel being cooled, while a portion of the reaction mixture is heated.3. The method according to claim 1, wherein the central portion of thereaction mixture is heated while the walls of the reaction vessel arebeing cooled.
 4. The method according to claim 1, wherein the reactionmixture is subjected to a centrifugal force exceeding 500×g.
 5. Themethod according to claim 1, wherein the reaction mixture is subjectedto a centrifugal force in the interval of about 500 to about 20,000×g.6. The method according to claim 1, wherein the reaction mixture issubjected to a centrifugal force in the interval of about 1,500 to about20,000×g.
 7. The method according to claim 1, wherein the reactionmixture is subjected to a centrifugal force in the interval of about5,000 to about 15,000×g.
 8. The method according to claim 1, wherein theheating of a subset of the reaction mixture is performed by a method ofheating chosen among an IR-source, and a microwave element.
 9. Themethod according to claim 2, wherein the cooling of the walls of thereaction vessel is performed by convection cooling or the use of acirculating cooling medium.
 10. A method for enhancing mass transportbetween a surface and the bulk of a solution, in contact with saidsurface, characterized in that said solution is placed in a vesselsuitable for centrifugation, and subjected to asymmetric heating,cooling and simultaneous centrifugation at conditions for creating aenhanced flow within said reaction mixture, wherein the asymmetricheating and cooling consists in the creation of a temperature differencebetween subsets of said reaction mixture and said flow ensurespractically total mixing and homogenisation of the reaction mixture. 11.The method according to claim 10, wherein the asymmetric heating andcooling consists of the walls of the reaction vessel being cooled, whilea portion of the reaction mixture is heated.
 12. The method according toclaim 10, wherein the central portion of the reaction mixture is heatedwhile the walls of the reaction vessel are being cooled.
 13. The methodaccording to claim 10, wherein the reaction mixture is subjected to acentrifugal force exceeding 500×g.
 14. The method according to claim 10,wherein the reaction mixture is subjected to a centrifugal force in theinterval of about 500 to about 20,000×g.
 15. The method according toclaim 10, wherein the reaction mixture is subjected to a centrifugalforce in the interval of about 1,500 to about 20,000×g.
 16. The methodaccording to claim 10, wherein the reaction mixture is subjected to acentrifugal force in the interval of about 5,000 to about 15,000×g. 17.The method according to claim 10, wherein the heating of a subset of thereaction mixture is performed by a method of heating chosen among anIR-source, and a microwave element.
 18. The method according to claim11, wherein the cooling of the walls of the reaction vessel is performedby convection cooling or the use of a circulating cooling medium.
 19. Amethod for performing chemical synthesis, comprising the method ofclaim
 1. 20. A method for performing chemical synthesis, comprising themethod of claim
 10. 21. A method according to claim 19, wherein thechemical synthesis is solid phase chemical synthesis.
 22. A method forperforming an assay, comprising the method of claim
 1. 23. A method forperforming an assay, comprising the method of claim
 10. 24. A methodaccording to claim 22, wherein the assay is a solid phase assay.
 25. Amethod for rapid temperature inactivation in chemical reactions, whereinthe reaction mixture is subjected to the method according to claim 1,the entire reaction mixture being rapidly brought to a temperaturesufficient to perform the desired inactivation.
 26. A device forperforming the method according to claim 1, wherein said device hasmeans for holding a vessel suitable for centrifugation, and means forsubjecting said vessel to asymmetric heating, cooling and simultaneouscentrifugation at conditions for creating an enhanced flow within saidreaction mixture, wherein said flow ensures practically total mixing andhomogenisation of the reaction mixture in said vessel.
 27. A device forperforming the method according to claim 10, wherein said device hasmeans for holding a vessel suitable for centrifugation, and means forsubjecting said vessel to asymmetric heating, cooling and simultaneouscentrifugation at conditions for creating an enhanced mass transportbetween the bulk of reaction mixture and the surface of said vessel,wherein said flow ensures that the entire bulk of the reaction mixturerepeatedly passes a defined location in the vessel.
 28. A reactionvessel (1), the configuration of which takes into account the flowpattern achieved in said vessel during asymmetric heating and coolingduring centrifugation.
 29. The reaction vessel (1) according to claim28, wherein said vessel has means (2, 3, 4, 5, 6, 7, 8, 9) capable ofinteraction with at least one component of the reaction mixture, placedat locations at the inner surface of the reaction vessel, correspondingto the collective down-ward flow or sink in said vessel.
 30. A reactionvessel (1) according to claim 28, wherein said vessel has a shape orfeatures (14) which aid in the positioning of said vessel so that theflow pattern achieved in said vessel during asymmetric heating andcooling thereof during centrifugation is accounted for, and so that thelocation of the collective down-ward flow or sink in said vessel can becontrolled.
 31. The reaction vessel according to claim 28, wherein saidreaction vessel is chosen among a centrifuge tube, a microtitre tube, awell in a microtitre plate, a volume defined in a device suitable forrotation, such as a platform for analysis in the CD-format.
 32. Thereaction vessel according to claim 29, wherein said means capable ofinteraction with at least one component of the reaction mixture aremeans chosen among mechanical means interacting with the flow, means fortransferring heat, means for guiding light or radiation, arrays, definedareas, dots or spots with a chemical or biochemical component whichinteracts with a component in the reaction mixture.
 33. An insert (9)for a reaction vessel (1) for use in a method according to claim 1,characterized in that said insert has means (10, 11, 12, 13) capable ofinteraction with at least one component of the reaction mixture, placedat locations at the surface of said insert, corresponding to thecollective down-ward flow or sink in said vessel, when said insert is inplace in said vessel.
 34. The insert according to claim 33, wherein saidmeans capable of interaction with at least one component of the reactionmixture are means chosen among mechanical means interacting with theflow, means for transferring heat, means for guiding light or radiation,arrays, defined areas, dots or spots with a chemical or biochemicalcomponent which interacts with a component in the reaction mixture. 35.A reaction vessel comprising, as a detachable or integrated partthereof, an insert according to claim 33.